Arsenic speciation analysis was performed in two human urine certified reference materials (NIES No. 18 and NIST SRM2670a) and three human urine control materials (Seronorm, Medisafe and Lyphocheck). The samples were diluted 1 + 3 prior to analysis by gradient elution anion or cation exchange high-performance liquid chromatography ( HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Nine arsenic species, including arsenic acid, arsenous acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, trimethylarsine oxide, dimethylarsinoylacetic acid, trimethylarsoniopropionate and dimethylarsinoylethanol, were determined in the urines. Additionally, several unknown arsenicals were detected. This is the first time that dimethylarsinoylacetic acid and trimethylarsoniopropionate have been reported in human urine. The sums of the species concentrations determined by the chromatographic approaches were identical with the reference values given for total arsenic. The obtained values for arsenobetaine and dimethylarsinic acid were identical with the values certified for the NIES No. 18 urine CRM. The speciation data presented here may be valuable for the quality assurance of analytical method development and surveys of arsenic in urine samples.